Multilevel systemic engineering of Bacillus licheniformis for efficient production of acetoin from lignocellulosic hydrolysates.

Bio-refining lignocellulosic resource offers a renewable and sustainable approach for producing biofuels and biochemicals. However, the conversion efficiency of lignocellulosic resource is still challenging due to the intrinsic inefficiency in co-utilization of xylose and glucose. In this study, the industrial bacterium Bacillus licheniformis was engineered for biorefining lignocellulosic resource to produce acetoin. First, adaptive evolution was conducted to improve acetoin tolerance, leading to a 19.6 % increase in acetoin production. Then, ARTP mutagenesis and 60Co-γ irradiation was carried out to enhance the production of acetoin, obtaining 73.0 g/L acetoin from glucose. Further, xylose uptake and xylose utilization pathway were rewired to facilitate the co-utilization of xylose and glucose, enabling the production of 60.6 g/L acetoin from glucose and xylose mixtures. Finally, this efficient cell factory was utilized for acetoin production from lignocellulosic hydrolysates with the highest titer of 68.3 g/L in fed-batch fermentation. This strategy described here holds great applied potential in the biorefinery of lignocellulose for the efficient synthesis of high-value chemicals.

Metabolic bottlenecks of Pseudomonas taiwanensis VLB120 during growth on d-xylose via the Weimberg pathway.

The microbial production of value-added chemicals from renewable feedstocks is an important step towards a sustainable, bio-based economy. Therefore, microbes need to efficiently utilize lignocellulosic biomass and its dominant constituents, such as d-xylose. Pseudomonas taiwanensis VLB120 assimilates d-xylose via the five-step Weimberg pathway. However, the knowledge about the metabolic constraints of the Weimberg pathway, i.e., its regulation, dynamics, and metabolite fluxes, is limited, which hampers the optimization and implementation of this pathway for bioprocesses. We characterized the Weimberg pathway activity of P. taiwanensis VLB120 in terms of biomass growth and the dynamics of pathway intermediates. In batch cultivations, we found excessive accumulation of the intermediates d-xylonolactone and d-xylonate, indicating bottlenecks in d-xylonolactone hydrolysis and d-xylonate uptake. Moreover, the intermediate accumulation was highly dependent on the concentration of d-xylose and the extracellular pH. To encounter the apparent bottlenecks, we identified and overexpressed two genes coding for putative endogenous xylonolactonases PVLB_05820 and PVLB_12345. Compared to the control strain, the overexpression of PVLB_12345 resulted in an increased growth rate and biomass generation of up to 30 % and 100 %, respectively. Next, d-xylonate accumulation was decreased by overexpressing two newly identified d-xylonate transporter genes, PVLB_18545 and gntP (PVLB_13665). Finally, we combined xylonolactonase overexpression with enhanced uptake of d-xylonate by knocking out the gntP repressor gene gntR (PVLB_13655) and increased the growth rate and biomass yield by 50 % and 24 % in stirred-tank bioreactors, respectively. Our study contributes to the fundamental knowledge of the Weimberg pathway in pseudomonads and demonstrates how to encounter the metabolic bottlenecks of the Weimberg pathway to advance strain developments and cell factory design for bioprocesses on renewable feedstocks.

Exploring xylose metabolism in non-conventional yeasts: kinetic characterization and product accumulation under different aeration conditions.

d-Xylose is a metabolizable carbon source for several non-Saccharomyces species, but not for native strains of S. cerevisiae. For the potential application of xylose-assimilating yeasts in biotechnological processes, a deeper understanding of pentose catabolism is needed. This work aimed to investigate the traits behind xylose utilization in diverse yeast species. The performance of 9 selected xylose-metabolizing yeast strains was evaluated and compared across 3 oxygenation conditions. Oxygenation diversely impacted growth, xylose consumption, and product accumulation. Xylose utilization by ethanol-producing species such as Spathaspora passalidarum and Scheffersomyces stipitis was less affected by oxygen restriction compared with other xylitol-accumulating species such as Meyerozyma guilliermondii, Naganishia liquefaciens, and Yamadazyma sp., for which increased aeration stimulated xylose assimilation considerably. Spathaspora passalidarum exhibited superior conversion of xylose to ethanol and showed the fastest growth and xylose consumption in all 3 conditions. By performing assays under identical conditions for all selected yeasts, we minimize bias in comparisons, providing valuable insight into xylose metabolism and facilitating the development of robust bioprocesses. ONE-SENTENCE SUMMARY: This work aims to expand the knowledge of xylose utilization in different yeast species, with a focus on how oxygenation impacts xylose assimilation.

Production of Optically Pure (S)-3-Hydroxy-γ-butyrolactone from d-Xylose Using Engineered Escherichia coli.

Biocatalysis has advantages in asymmetric synthesis due to the excellent stereoselectivity of enzymes. The present study established an efficient biosynthesis pathway for optically pure (S)-3-hydroxy-γ-butyrolactone [(S)-3HγBL] production using engineered Escherichia coli. We mimicked the 1,2,4-butanetriol biosynthesis route and constructed a five-step pathway consisting of d-xylose dehydrogenase, d-xylonolactonase, d-xylonate dehydratase, 2-keto acid decarboxylase, and aldehyde dehydrogenase. The engineered strain harboring the five enzymes could convert d-xylose to 3HγBL with glycerol as the carbon source. Stereochemical analysis by chiral GC proved that the microbially synthesized product was a single isomer, and the enantiomeric excess (ee) value reached 99.3%. (S)-3HγBL production was further enhanced by disrupting the branched pathways responsible for d-xylose uptake and intermediate reduction. Fed-batch fermentation of the best engineered strain showed the highest (S)-3HγBL titer of 3.5 g/L. The volumetric productivity and molar yield of (S)-3HγBL on d-xylose reached 50.6 mg/(L·h) and 52.1%, respectively. The final fermentation product was extracted, purified, and confirmed by NMR. This process utilized renewable d-xylose as the feedstock and offered an alternative approach for the production of the valuable chemical.

Construction of prokaryotic nanocompartment in Yarrowia lipolytica to assist phloroglucinol production.

Phloroglucinol is an important chemical intermediate which has been tentatively produced by engineered bacteria. However, its biosynthesis in industry is limited due to its natural antibacterial activity. Our study firstly selected Yarrowia lipolytica as the chassis strain, which was verified to be tolerable to phloroglucinol. Then the gene of type III polyketone synthase PhlD (the key biosynthetic gene) was overexpressed to facilitate phloroglucinol production with a concentration of 107.4 mg/L. Furthermore, we introduced the prokaryotic nanocompartment to assist the intracellular catalytic activity. The results showed that the concentration of phloroglucinol was increased by about 2.5 times, indicating this multifunctional nanocompartment is orthogonal to the physiological activities of Y. lipolytica. Additionally, fermentations with xylose and lignocellulosic hydrolysates as the carbon source were performed with the engineered Y. lipolytica, resulting in a total concentration of 580.2 mg/L and 328.9 mg/L, respectively. These findings revealed the potential of Y. lipolytica in phloroglucinol production and provided an effective nanocompartment strategy to improve the catalytic activity of the enzyme for boosting phloroglucinol production. KEY POINTS: • The first time to select and use Y. lipolytica to produce phloroglucinol. • Successful construction of prokaryotic nanocompartment in Y. lipolytica to increase production of phloroglucinol. • Lignocellulose hydrolysate is used as a substrate in fermentation.

Metabolic Engineering of Pichia pastoris for the Production of Triacetic Acid Lactone.

Triacetic acid lactone (TAL) is a promising renewable platform polyketide with broad biotechnological applications. In this study, we constructed an engineered Pichia pastoris strain for the production of TAL. We first introduced a heterologous TAL biosynthetic pathway by integrating the 2-pyrone synthase encoding gene from Gerbera hybrida (Gh2PS). We then removed the rate-limiting step of TAL synthesis by introducing the posttranslational regulation-free acetyl-CoA carboxylase mutant encoding gene from S. cerevisiae (ScACC1*) and increasing the copy number of Gh2PS. Finally, to enhance intracellular acetyl-CoA supply, we focused on the introduction of the phosphoketolase/phosphotransacetylase pathway (PK pathway). To direct more carbon flux towards the PK pathway for acetyl-CoA generation, we combined it with a heterologous xylose utilization pathway or endogenous methanol utilization pathway. The combination of the PK pathway with the xylose utilization pathway resulted in the production of 825.6 mg/L TAL in minimal medium with xylose as the sole carbon source, with a TAL yield of 0.041 g/g xylose. This is the first report on TAL biosynthesis in P. pastoris and its direct synthesis from methanol. The present study suggests potential applications in improving the intracellular pool of acetyl-CoA and provides a basis for the construction of efficient cell factories for the production of acetyl-CoA derived compounds.

The growth and lipid accumulation of Scenedesmus quadricauda under nitrogen starvation stress during xylose mixotrophic/heterotrophic cultivation.

In order to conquer the block of high cost and low yields which limit to realize the commercialization of microalgal biodiesel, the mixotrophic and heterotrophic cultivation of Scenedesmus quadricauda FACHB-1297 fed on xylose was separately studied employing six forms of media: phosphorus sufficient, phosphorus restricted, and phosphorus starvation were combined with nitrogen sufficient and nitrogen starvation conditions. The maximum lipid content (about 41% of dry weight) was obtained on the 5th day (heterotrophic cultivation) and 8th day (mixotrophic cultivation) under the nitrogen starved and phosphorus sufficient (N0&P) conditions, which was about twofold in comparison to the final lipid content on the sufficient nitrogen condition (control). Under mixotrophic and heterotrophic modes, the highest lipid production was achieved in the N0&P trial, with the value of 274.96 mg/L and 193.77 mg/L, respectively. Xylose utilization rate of 30-96% under heterotrophic modes was apparently higher than that of 20-50% in mixotrophic modes. In contrast, phosphorus uptake rate of 100% under mixotrophic cultivation was significantly more than that of 60-90% in heterotrophic cultivation. Furthermore, under the condition of heterotrophic cultivation using xylose as a carbon source, the phosphorus had a positive impact on microalgae cell synthesis and the lipid content enhanced with the augmentation in phosphorus concentrations. We suggested that sufficient phosphorus should be supplied for obtaining higher microalgal lipid production in the lack of nitrogen under xylose heterotrophic/mixotrophic condition. This was a highly effective way to obtain efficient microalgae lipid production.

Lipid production from lignocellulosic biomass using an engineered Yarrowia lipolytica strain.

BACKGROUND: The utilization of industrial wastes as feedstock in microbial-based processes is a one of the high-potential approach for the development of sustainable, environmentally beneficial and valuable bioproduction, inter alia, lipids. Rye straw hydrolysate, a possible renewable carbon source for bioconversion, contains a large amount of xylose, inaccessible to the wild-type Yarrowia lipolytica strains. Although these oleaginous yeasts possesses all crucial genes for xylose utilization, it is necessary to induce their metabolic pathway for efficient growth on xylose and mixed sugars from agricultural wastes. Either way, biotechnological production of single cell oils (SCO) from lignocellulosic hydrolysate requires yeast genome modification or adaptation to a suboptimal environment. RESULTS: The presented Y. lipolytica strain was developed using minimal genome modification-overexpression of endogenous xylitol dehydrogenase (XDH) and xylulose kinase (XK) genes was sufficient to allow yeast to grow on xylose as a sole carbon source. Diacylglycerol acyltransferase (DGA1) expression remained stable and provided lipid overproduction. Obtained an engineered Y. lipolytica strain produced 5.51 g/L biomass and 2.19 g/L lipids from nitrogen-supplemented rye straw hydrolysate, which represents an increase of 64% and an almost 10 times higher level, respectively, compared to the wild type (WT) strain. Glucose and xylose were depleted after 120 h of fermentation. No increase in byproducts such as xylitol was observed. CONCLUSIONS: Xylose-rich rye straw hydrolysate was exploited efficiently for the benefit of production of lipids. This study indicates that it is possible to fine-tune a newly strain with as minimally genetic changes as possible by adjusting to an unfavorable environment, thus limiting multi-level genome modification. It is documented here the use of Y. lipolytica as a microbial cell factory for lipid synthesis from rye straw hydrolysate as a low-cost feedstock.

Deletion of NGG1 in a recombinant Saccharomyces cerevisiae improved xylose utilization and affected transcription of genes related to amino acid metabolism.

Production of biofuels and biochemicals from xylose using yeast cell factory is of great interest for lignocellulosic biorefinery. Our previous studies revealed that a natural yeast isolate Saccharomyces cerevisiae YB-2625 has superior xylose-fermenting ability. Through integrative omics analysis, NGG1, which encodes a transcription regulator as well as a subunit of chromatin modifying histone acetyltransferase complexes was revealed to regulate xylose metabolism. Deletion of NGG1 in S. cerevisiae YRH396h, which is the haploid version of the recombinant yeast using S. cerevisiae YB-2625 as the host strain, improved xylose consumption by 28.6%. Comparative transcriptome analysis revealed that NGG1 deletion down-regulated genes related to mitochondrial function, TCA cycle, ATP biosynthesis, respiration, as well as NADH generation. In addition, the NGG1 deletion mutant also showed transcriptional changes in amino acid biosynthesis genes. Further analysis of intracellular amino acid content confirmed the effect of NGG1 on amino acid accumulation during xylose utilization. Our results indicated that NGG1 is one of the core nodes for coordinated regulation of carbon and nitrogen metabolism in the recombinant S. cerevisiae. This work reveals novel function of Ngg1p in yeast metabolism and provides basis for developing robust yeast strains to produce ethanol and biochemicals using lignocellulosic biomass.

A novel fermentation strategy for efficient xylose utilization and microbial lipid production in lignocellulosic hydrolysate.

The sugar utilization efficiency and the tolerance of microorganism to inhibitors are essential for lipid production from lignocellulosic biomass. In this study, the sugar consumption and inhibitor tolerance characteristics of Trichosporon dermatis 32,903 were investigated. The results showed that the lipid yield on xylose was much lower than that on glucose, while these substrates exhibited comparative efficiency for cell growth. High inoculum size improved the tolerance of T. dermatis 32,903 to inhibitors. Based on these characteristics, sugar-targeted-utilization and cyclic fermentation strategy was developed. The tolerance of high inoculum size to inhibitors was utilized, glucose was targeted for lipid fermentation and xylose was targeted for cell growth. As a result, the lipid production efficiency was greatly enhanced. The lipid titer in hydrolysate of DLCA (Densifying Lignocellulosic biomass with Chemicals followed by Autoclave) pretreated rice straw was improved to as high as 38.4 g/L with lipid yield of 0.207 g/g consumed sugar.

Engineering a Xylose-Utilizing Synechococcus elongatus UTEX 2973 Chassis for 3-Hydroxypropionic Acid Biosynthesis under Photomixotrophic Conditions.

Photomixotrophic cultivation of cyanobacteria is considered a promising strategy to achieve both high cell density and product accumulation, since cyanobacteria can obtain carbon and energy sources from organic matter in addition to those obtained from CO2 and sunlight. Acetyl coenzyme A (acetyl-CoA) is a key precursor used for the biosynthesis of a wide variety of important value-added chemicals. However, the acetyl-CoA content in cyanobacteria is typically low under photomixotrophic conditions, which limits the productivity of the derived chemicals. In this study, a xylose utilization pathway from Escherichia coli was first engineered into fast-growing Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 2973), enabling the xylose based photomixotrophy. Metabolomics analysis of the engineered strain showed that the utilization of xylose enhanced the carbon flow to the oxidative pentose phosphate (OPP) pathway, along with an increase in the intracellular abundance of metabolites such as fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (FBP), ribose-5-phosphate (R5P), erythrose-4-phosphate (E4P), and glyceraldehyde-3-phosphate (G3P). Then, the native glycolytic pathway was rewired via heterologous phosphoketolase (Pkt) gene expression, combined with phosphofructokinase (Pfk) gene knockout and fructose-1,6-bisphosphatase (Fbp) gene overexpression, to drive more carbon flux from xylose to acetyl-CoA. Finally, a heterologous 3-hydroxypropionic acid (3-HP) biosynthetic pathway was introduced. The results showed that 3-HP biosynthesis was improved by up to approximately 4.1-fold (from 22.5 mg/L to 91.3 mg/L) compared with the engineered strain without a rewired metabolism under photomixotrophic conditions and up to approximately 14-fold compared with the strain under photoautotrophic conditions. Using 3-HP as a "proof-of-molecule", our results demonstrated that this strategy could be applied to improve the intracellular pool of acetyl-CoA for the photomixotrophic production of value-added chemicals that require acetyl-CoA as a precursor in a cyanobacterial chassis.

Assessing the potential of Schizochytrium sp. HX-308 for microbial lipids production from corn stover hydrolysate.

Schizochytrium sp. has received increasing attention as promising commercial resource for the sustainable production of lipids, due to their fast growth rate and high lipid content. However, the price of glucose represents a significant proportion of the total substrate cost. Therefore, in this study, the lignocellulosic hydrolysate of corn stover hydrolysate (CSH) was used as low-cost culture medium to replace glucose in Schizochytrium sp. fermentation. When Schizochytrium sp. HX-308 was fermented with 20% glucose from CSH and 80% of glucose from pure glucose, the lipid production reached 21.2 g L-1 , which is lower than that of using 100% of pure glucose. However, the shifts of fatty acid composition indicated that CSH has great potential to enhance the percentage of polyunsaturated fatty acids (PUFAs) in total lipids. However, as the second largest carbon source in CSH, xylose was not utilized by the Schizochytrium sp. HX-308, and further analysis showed that probably because it does not possess a functional xylulose kinase. In addition, the degradation products in lignocellulosic hydrolysate have a strong inhibitory effect on cell growth, so it is necessary to investigate the tolerance of Schizochytrium sp. HX-308 to degradation products. Here, the effects of five typical degradation products on the growth and lipid synthesis were further investigated. Schizochytrium sp. HX-308 showed good tolerance to furan derivatives and organic acids, but low tolerance to phenolic compounds. Furthermore, in order to improve the lipid accumulation using CSH, the two-stage fermentation strategy was developed, resulting in a 54.8% increase compared to that of the one-stage strategy. In summary, this study provides a reference for further fermentation engineering with cheap lignocellulosic biomass as substrate.

Development and characterization of efficient xylose utilization strains of Zymomonas mobilis.

BACKGROUND: Efficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level. RESULTS: Heterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment. CONCLUSIONS: This study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.

Improved glucose and xylose co-utilization by overexpression of xylose isomerase and/or xylulokinase genes in oleaginous fungus Mucor circinelloides.

Most of the oleaginous microorganisms cannot assimilate xylose in the presence of glucose, which is the major bottleneck in the bioconversion of lignocellulose to biodiesel. Our present study revealed that overexpression of xylose isomerase (XI) gene xylA or xylulokinase (XK) gene xks1 increased the xylose consumption by 25 to 37% and enhanced the lipid content by 8 to 28% during co-fermentation of glucose and xylose. In xylA overexpressing strain Mc-XI, the activity of XI was 1.8-fold higher and the mRNA level of xylA at 24 h and 48 h was 11- and 13-fold higher than that of the control, respectively. In xks1 overexpressing strain Mc-XK, the mRNA level of xks1 was 4- to 11-fold of that of the control strain and the highest XK activity of 950 nmol min-1 mg-1 at 72 h which was 2-fold higher than that of the control. Additionally, expression of a translational fusion of xylA and xks1 further enhanced the xylose utilization rate by 45%. Our results indicated that overexpression of xylA and/or xks1 is a promising strategy to improve the xylose and glucose co-utilization, alleviate the glucose repression, and produce lipid from lignocellulosic biomass in the oleaginous fungus M. circinelloides. KEY POINTS: • Overexpressing xylA or xks1 increased the xylose consumption and the lipid content. • The xylose isomerase activity and the xylA mRNA level were enhanced in strain Mc-XI. • Co-expression of xylA and xks1 further enhanced the xylose utilization rate by 45%.

Tolerance of Yarrowia lipolytica to inhibitors commonly found in lignocellulosic hydrolysates.

BACKGROUND: Lignocellulosic material is a suitable renewable carbon and energy source for microbial cell factories, such as Yarrowia lipolytica. To be accessible for microorganisms, the constituent sugars need to be released in a hydrolysis step, which as a side effect leads to the formation of various inhibitory compounds. However, the effects of these inhibitory compounds on the growth of Y. lipolytica have not been thoroughly investigated. RESULTS: Here we show the individual and combined effect of six inhibitors from three major inhibitor groups on the growth of Y. lipolytica. We engineered a xylose consuming strain by overexpressing the three native genes XR, XDH, and XK and found that the inhibitor tolerance of Y. lipolytica is similar in glucose and in xylose. Aromatic compounds could be tolerated at high concentrations, while furfural linearly increased the lag phase of the cultivation, and hydroxymethylfurfural only inhibited growth partially. The furfural induced increase in lag phase can be overcome by an increased volume of inoculum. Formic acid only affected growth at concentrations above 25 mM. In a synthetic hydrolysate, formic acid, furfural, and coniferyl aldehyde were identified as the major growth inhibitors. CONCLUSION: We showed the individual and combined effect of inhibitors found in hydrolysate on the growth of Y. lipolytica. Our study improves understanding of the growth limiting inhibitors found in hydrolysate and enables a more targeted engineering approach to increase the inhibitor tolerance of Y. lipolytica. This will help to improve the usage of Y. lipolytica as a sustainable microbial cell factory.

Production of poly-γ-glutamic acid (γ-PGA) from xylose-glucose mixtures by Bacillus amyloliquefaciens C1.

Due to the promising applications, the demand to enhance poly-γ-glutamic acid (γ-PGA) production while decreasing the cost has increased in the past decade. Here, xylose/glucose mixture and corncob hydrolysate (CCH) was evaluated as alternatives for γ-PGA production by Bacillus amyloliquefaciens C1. Although both have been validated to support cell growth, glucose and xylose were not simutaneously consumed and exhibited a diauxic growth pattern due to carbon catabolite repression (CCR) in B. amyloliquefaciens C1, while the enhanced transcription of araE alleviated the xylose transport bottleneck across a cellular membrane. Additionally, the xyl operon (xylA and xylB), which was responsible for xylose metabolism, was strongly induced by xylose at the transcriptional level. When cultured in a mixed medium, xylR was sharply induced to 3.39-folds during the first 8-h while reduced to the base level similar to that in xylose medium. Finally, pre-treated CCH mainly contained a mixture of glucose and xylose was employed for γ-PGA fermentation, which obtained a final concentration of 6.56 ± 0.27 g/L. Although the glucose utilization rate (84.91 ± 1.81%) was lower than that with chemical substrates, the xylose utilization rate (43.41 ± 2.14%) and the sodium glutamate conversion rate (77.22%) of CCH were acceptable. Our study provided a promising approach for the green production of γ-PGA from lignocellulosic biomass and circumvent excessive non-food usage of glucose.

Complete genome sequence of Paenibacillus xylanexedens PAMC 22703, a xylan-degrading bacterium.

Paenibacillus is widely distributed in various environments and has the potential for use as a biotechnological agent in industrial processes. Here, we report the complete genome sequence of the marine bacterium, Paenibacillus xylanexedens PAMC 22703, which utilizes xylan. The P. xylanexedens PAMC 22703 strain was isolated from marine sediments. P. xylanexedens PAMC 22703 utilizes xylan as a carbon source to grow. The genome sequence clarified that this strain possesses genes for utilizing xylan. The complete genome sequence contained one chromosome (7,053,622 bp with 46.0% GC content) and one plasmid (44,617 bp with 44.1% C + G content). The genome harbored genes that fully deploy the xylan assimilation pathway. The complete genome sequence of P. xylanexedens PAMC 22703 would prove useful in acquiring information for its application with xylan in various industries.

Metabolically engineering of Yarrowia lipolytica for the biosynthesis of naringenin from a mixture of glucose and xylose.

Xylose-inducible modules simultaneously expressing xylose utilization and naringenin biosynthesis pathways were developed in Yarrowia lipolytica to produce naringenin from a mixture of glucose and xylose. The naringenin synthetic pathway was constructed using a constitutive expression to yield 239.1 ± 5.1 mg/L naringenin. Furthermore, the introduction of an inducible pathway realized the dual function of xylose as a substrate and synthetic inducer, which coupled the xylose utilization with naringenin biosynthesis and increased production. Interestingly, the simultaneous enhancement of xylose reductase and xylose transporter expression along with that of xylitol dehydrogenase and xylulokinase can further improve the xylose utilization ability of Y. lipolytica. As expected, xylose-inducible synthesis of naringenin could achieved a titer of 715.3 ± 12.8 mg/L through the shake-flask cultivation level. Therefore, xylose-induced activation of both the xylose utilization and product biosynthesis pathway is considered to be an effective strategy for the biosynthesis of xylose-derived chemicals in yeast.

Production of bioethanol and xylitol from non-detoxified corn cob via a two-stage fermentation strategy.

A novel two-stage fermentation strategy was applied to produce xylitol and ethanol from the whole acid-pretreated corn cob slurry. The acid-pretreated corn cob was used without filtration and detoxification by the two-stage fermentation with the robust Kluyveromyces marxianus CICC 1727-5. In the first stage, xylose in the slurry after dilute acid pretreatment of lignocellulosic biomass was used to produce xylitol under micro-aeration conditions. In the second stage, simultaneous saccharification fermentation was carried out, and the ethanol was produced from glucose releasing from the solid. Important parameters, such as aeration rate, cellulase loading during xylose utilization and SSF fermentation were studied for best performance. The two-stage fermentation strategy removed the inhibition of glucose on xylose, and little xylose was left in the fermentation broth. Under the optimized condition, the maximum ethanol and xylitol concentration were 52 g/L and 24.2 g/L corresponding to the yield of 0.41 g/g and 0.82 g/g, respectively.

Improvement in D-xylose utilization and isobutanol production in S. cerevisiae by adaptive laboratory evolution and rational engineering.

As the effects of climate change become apparent, metabolic engineers and synthetic biologists are exploring sustainable sources for transportation fuels. The design and engineering of microorganisms to produce gasoline, diesel, and jet fuel compounds from renewable feedstocks can significantly reduce our dependence on fossil fuels as well as lower the emissions of greenhouse gases. Over the past 2 decades, a considerable amount of work has led to the development of microbial strains for the production of advanced fuel compounds from both C5 and C6 sugars. In this work, we combined two strategies-adaptive laboratory evolution and rational metabolic engineering-to improve the yeast Saccharomyces cerevisiae's ability to utilize D-xylose, a major C5 sugar in biomass, and produce the advanced biofuel isobutanol. Whole genome resequencing of several evolved strains followed by reverse engineering identified two single nucleotide mutations, one in CCR4 and another in TIF1, that improved the yeast's specific growth rate by 23% and 14%, respectively. Neither one of these genes has previously been implicated to play a role in utilization of D-xylose. Fine-tuning the expression levels of the bottleneck enzymes in the isobutanol pathway further improved the evolved strain's isobutanol titer to 92.9 ± 4.4 mg/L (specific isobutanol production of 50.2 ± 2.6 mg/g DCW), a 90% improvement in titer and a 110% improvement in specific production over the non-evolved strain. We hope that our work will set the stage for an economic route to the advanced biofuel isobutanol and enable efficient utilization of xylose-containing biomass.