Rethinking Biosynthesis of Aclacinomycin A.

Aclacinomycin A (ACM-A) is an anthracycline antitumor agent widely used in clinical practice. The current industrial production of ACM-A relies primarily on chemical synthesis and microbial fermentation. However, chemical synthesis involves multiple reactions which give rise to high production costs and environmental pollution. Microbial fermentation is a sustainable strategy, yet the current fermentation yield is too low to satisfy market demand. Hence, strain improvement is highly desirable, and tremendous endeavors have been made to decipher biosynthesis pathways and modify key enzymes. In this review, we comprehensively describe the reported biosynthesis pathways, key enzymes, and, especially, catalytic mechanisms. In addition, we come up with strategies to uncover unknown enzymes and improve the activities of rate-limiting enzymes. Overall, this review aims to provide valuable insights for complete biosynthesis of ACM-A.

CRISPR-Cas13 technology portfolio and alliance with other genetic tools.

The exploitation of CRISPR-Cas systems especially CRISPR-Cas9 has led to radical advances in genome editing, gene activation, gene repression, protein imaging, and beyond. However, these applications are limited to targeting of DNA rather than RNA. CRISPR-Cas13 is the first reported CRISPR system targeting RNA and thus opens a new avenue for transcription regulation. While a plethora of reviews have systematically documented CRISPR-Cas9 toolbox, this review focuses on CRISPR-Cas13 family, covering aspects of classification, structures, response to foreign invaders, and genetic toolbox. In particular, we compare CRISPR-Cas13 with other RNA regulation tools such as RNA interference and antisense RNA technology to ponder the possibility of combining them to engineer hierarchical regulatory networks fulfilling novel functions. Lastly, we summarize the wide applications of CRISPR-Cas13 toolbox and the status quo that requires amelioration. Overall, this review charts a landscape of CRISPR-Cas13 technology portfolio to provide insights for gene regulation, metabolic engineering and synthetic biology.

Genomic landscapes of bacterial transposons and their applications in strain improvement.

Transposons are mobile genetic elements that can give rise to gene mutation and genome rearrangement. Due to their mobility, transposons have been exploited as genetic tools for modification of plants, animals, and microbes. Although a plethora of reviews have summarized families of transposons, the transposons from fermentation bacteria have not been systematically documented, which thereby constrain the exploitation for metabolic engineering and synthetic biology purposes. In this review, we summarize the transposons from the most used fermentation bacteria including Escherichia coli, Bacillus subtilis, Lactococcus lactis, Corynebacterium glutamicum, Klebsiella pneumoniae, and Zymomonas mobilis by literature retrieval and data mining from GenBank and KEGG. We also outline the state-of-the-art advances in basic research and industrial applications especially when allied with other genetic tools. Overall, this review aims to provide valuable insights for transposon-mediated strain improvement. KEY POINTS: • The transposons from the most-used fermentation bacteria are systematically summarized. • The applications of transposons in strain improvement are comprehensively reviewed.

Delineating biosynthesis of Huperzine A, A plant-derived medicine for the treatment of Alzheimer's disease.

Huperzine A (HupA) is a plant-derived lycopodium alkaloid used for the treatment of Alzheimer's disease due to its inhibition against acetylcholinesterase. Currently, industrial production of HupA relies primarily on direct extraction from Huperzia serrate, a perennial herbaceous plant. However, this strategy cannot satisfy the increasing demand for HupA due to scarcity of H. serrate whose growth is quite slow. Pathway engineering has emerged as a novel strategy for the production of HupA. Unfortunately, the biosynthesis mechanism of HupA has not been well documented. In this review, we summarize not only the methods for plant extraction and chemical synthesis but also state-of-the-art advances in biosynthesis of HupA, including synthetic pathways, key enzymes, and especially catalytic mechanisms. Overall, this review aims to provide valuable insights for complete biosynthesis of Hup A.

Engineering Glucose-to-Glycerol Pathway in Klebsiella pneumoniae and Boosting 3-Hydroxypropionic Acid Production Through CRISPR Interference.

The recent decline of the international biodiesel industry has led to decreased production and therefore increased the price of glycerol, which is a major by-product of biodiesel but a substrate for production of 3-hydroxypropionic acid (3-HP), that is, glycerol as a feedstock has no advantage over glucose in price. Hence, we engineered glucose to the glycerol pathway and improved 3-HP production by CRISPR interference (CRISPRi). To begin with, we cloned the genes encoding glycerol 3-phosphate dehydrogenase (gpd1) and glycerol 3-phosphatase (gpp2) from Saccharomyces cerevisiae, which jointly catalyze glucose into glycerol. The genes gpd1 and gpp2 were co-expressed in K. pneumoniae with the dCas9 gene integrated in genome, and this recombinant strain produced 2 g/L glycerol in the shake flask. To minimize the glucose consumption by competing pathways including the EMP pathway, glycerol oxidation pathway, and by-products pathways, we developed an CRISPRi system in aforementioned recombinant K. pneumoniae strain to inhibit the expression of the glyceraldehyde-3-phosphate dehydrogenase gene (gapA) and 2,3-butanediol production gene (budA), resulting in a bi-functional strain harboring both glucose-to-glycerol pathway and CRISPRi system. Reverse transcription and quantitative PCR (RT-qPCR) results showed that this engineered CRISPRi system transcriptionally inhibited gapA and budA by 82% and 24%, respectively. In shake flask cultivation, this bi-functional strain produced 2.8 g/L glycerol using glucose as the carbon source, which was 46.6% increase compared to the strain without the engineered CRISPRi system. Moreover, this bi-functional strain produced 0.78 g/L 3-HP using glucose as the sole carbon source. In fed-batch cultivation, this bi-functional strain produced 1.77 g/L 3-HP. This study provides insights for co-utilization of distinct carbon sources.

Charting the landscape of RNA polymerases to unleash their potential in strain improvement.

One major mission of microbial cell factory is overproduction of desired chemicals. To this end, it is necessary to orchestrate enzymes that affect metabolic fluxes. However, only modification of a small number of enzymes in most cases cannot maximize desired metabolites, and global regulation is required. Of myriad enzymes influencing global regulation, RNA polymerase (RNAP) may be the most versatile enzyme in biological realm because it not only serves as the workhorse of central dogma but also participates in a plethora of biochemical events. In fact, recent years have witnessed extensive exploitation of RNAPs for phenotypic engineering. While a few impressive reviews showcase the structures and functionalities of RNAPs, this review not only summarizes the state-of-the-art advance in the structures of RNAPs but also points out their enormous potentials in metabolic engineering and synthetic biology. This review aims to provide valuable insights for strain improvement.

Biosynthesis pathways and strategies for improving 3-hydroxypropionic acid production in bacteria.

3-Hydroxypropionic acid (3-HP) represents an economically important platform compound from which a panel of bulk chemicals can be derived. Compared with petroleum-dependent chemical synthesis, bioproduction of 3-HP has attracted more attention due to utilization of renewable biomass. This review outlines bacterial production of 3-HP, covering aspects of host strains (e.g., Escherichia coli and Klebsiella pneumoniae), metabolic pathways, key enzymes, and hurdles hindering high-level production. Inspired by the state-of-the-art advances in metabolic engineering and synthetic biology, we come up with protocols to overcome the hurdles constraining 3-HP production. The protocols range from rewiring of metabolic networks, alleviation of metabolite toxicity, to dynamic control of cell size and density. Especially, this review highlights the substantial contribution of microbial growth to 3-HP production, as we recognize the synchronization between cell growth and 3-HP formation. Accordingly, we summarize the following growth-promoting strategies: (i) optimization of fermentation conditions; (ii) construction of gene circuits to alleviate feedback inhibition; (iii) recruitment of RNA polymerases to overexpress key enzymes which in turn boost cell growth and 3-HP production. Lastly, we propose metabolic engineering approaches to simplify downstream separation and purification. Overall, this review aims to portray a picture of bacterial production of 3-HP.

[Pathway design and key enzyme analysis of diosgenin biosynthesis].

As a naturally occurring steroid sapogenin, diosgenin acts as the precursor of hundreds of steroid medicines, and thereby has important medicinal value. Currently, industrial production of diosgenin relies primarily on chemical extraction from plant materials. Clearly, this strategy shows drawbacks of excessive reliance on plant materials and farmland as well as environment pollution. Due to development of metabolic engineering and synthetic biology, bio-production of diosgenin has garnered plenty of attention. Although the biosynthetic pathways of diosgenin have not been completely identified, in this review, we outline the identified biosynthetic pathways and key enzymes. In particular, we suggest heterologous biosynthesis of diosgenin in Saccharomyces cerevisiae. Overall, this review aims to provide valuable insights for future complete biosynthesis of diosgenin.

Switching metabolic flux by engineering tryptophan operon-assisted CRISPR interference system in Klebsiella pneumoniae.

One grand challenge for bioproduction of desired metabolites is how to coordinate cell growth and product synthesis. Here we report that a tryptophan operon-assisted CRISPR interference (CRISPRi) system can switch glycerol oxidation and reduction pathways in Klebsiella pneumoniae, whereby the oxidation pathway provides energy to sustain growth, and the reduction pathway generates 1,3-propanediol and 3-hydroxypropionic acid (3-HP), two economically important chemicals. Reverse transcription and quantitative PCR (RT-qPCR) showed that this CRISPRi-dependent switch affected the expression of glycerol metabolism-related genes and in turn improved 3-HP production. In shake-flask cultivation, the strain coexpressing dCas9-sgRNA and PuuC (an aldehyde dehydrogenase native to K. pneumoniae for 3-HP biosynthesis) produced 3.6 g/L 3-HP, which was 1.62 times that of the strain only overexpressing PuuC. In a 5 L bioreactor, this CRISPRi strain produced 58.9 g/L 3-HP. When circulation feeding was implemented to alleviate metabolic stress, biomass was substantially improved and 88.8 g/L 3-HP was produced. These results indicated that this CRISPRi-dependent switch can efficiently reconcile biomass formation and 3-HP biosynthesis. Furthermore, this is the first report of coupling CRISPRi system with trp operon, and this architecture holds huge potential in regulating gene expression and allocating metabolic flux.

A rapid liquid chromatography-electrospray ionization-ion mobility spectrometry method for monitoring nine representative metabolites in the seedlings of cucumber and wheat.

A fast and convenient high-performance liquid chromatography-electrospray ionization-ion mobility spectrometry method was developed to determine nine representative metabolites in the seedlings of cucumber and wheat. The analytical conditions were obtained by optimizing the parameters of high-performance liquid chromatography and ion mobility spectrometry. Briefly, acetonitrile-0.1% formic acid solution was selected as the mobile phase for gradient elution at a flow velocity of 0.4 mL/min. Under negative electrospray ionization mode, spray voltage of ion mobility spectrometry was 4.5 kV, and drift tube temperature was set at 90°C. The metabolites from seedling leaves were extracted using 80% acetonitrile as the solvent at 4°C for 12 h. Results showed that under soilless culture conditions, the contents of maltose, citric acid, and p-hydroxybenzoic acid in the seedlings of cucumber and wheat were reduced by low concentration of itaconic acid, succinic acid, and citric acid. Importantly, this analytical approach demonstrated high sensitivity, good linear response, and high selectivity. The lowest limit of detection was 0.004 µg for p-hydroxybenzoic acid. Overall, this high-performance liquid chromatography-electrospray ionization-ion mobility spectrometry method is sensitive and efficient for rapid separation and identification of plant metabolites.

Intensifying niacin-based biosynthesis of NAD+ to enhance 3-hydroxypropionic acid production in Klebsiella pneumoniae.

OBJECTIVE: Glycerol-based biosynthesis of 3-hydroxypropionic acid (3-HP) in Klebsiella pneumoniae involves two reactions: glycerol conversion to 3-hydroxypropionaldehyde (3-HPA) by glycerol dehydratase, and 3-HPA conversion to 3-HP by aldehyde dehydrogenase (ALDH). The ALDH catalysis consumes a lot of cofactor nicotinamide adenine dinucleotide (NAD+), which constrains 3-HP production. RESULTS: Here we report that intensifying niacin-based biosynthesis of NAD+ can substantially enhance 3-HP production. We constructed tac promoter-driven NAD+ synthesis pathway in K. pneumoniae. The strain only overexpressing nicotinate phosphoribosyltransferase (PncB) showed 14.24% increase in the production of NAD+ relative to the stain harboring an empty vector. When PncB was coexpressed with PuuC (one of native ALDHs), the recombinant strain exhibited increased ALDH activity but slightly reduced 3-HP production due to plasmid burden. When 30 mg niacin l-1 (a substrate for biosynthesis of NAD+) was added into shake flask, the strain produced 0.55 g 3-HP l-1, which was 2.75 times that of the control. In a 5-L bioreactor, replenishment of niacin led to 36.43% increase of 3-HP production. CONCLUSIONS: These results indicated that intensifying niacin-based biosynthesis of NAD+ boosts 3-HP production.

CRISPR interference-guided modulation of glucose pathways to boost aconitic acid production in Escherichia coli.

BACKGROUND: One major mission of microbial breeding is high-level production of desired metabolites. Overproduction of intermediate metabolites in core pathways is challenging as it may impair cell growth and viability. RESULTS: Here we report that aconitic acid, an intermediate metabolite in tricarboxylic acid (TCA) cycle, can be overproduced by an engineered CRISPR interference (CRISPRi) system in Escherichia coli. This CRISPRi system was designed to simultaneously target pyruvate kinase (PK) and isocitrate dehydrogenase (IDH), two enzymes in glycolytic pathway and TCA cycle, respectively. Reverse transcription and quantitative PCR and enzyme activity assays showed that this engineered CRISPRi system significantly repressed the genes encoding IDH and PK, resulting in simultaneous reduction in the activities of IDH and PK. In shake-flask and fed-batch cultivation, this CRISPRi strain produced 60-fold (362.80 ± 22.05 mg/L) and 15-fold (623.80 ± 20.05 mg/L) of aconitic acid relative to the control strain, respectively. In addition, this two-target CRISPRi strain maintained low levels of acetate and lactate, two problematic byproducts. CONCLUSIONS: This work demonstrates that CRISPRi system can improve aconitic acid production by coordinating glycolysis and TCA cycle. This study provides insights for high-level production of the intermediate metabolites in central pathways.

Overview of Alternaria alternata Membrane Proteins.

Alternaria species are mainly saprophytic fungi, but some pathotypes of Alternaria alternata infect economically important plants including cereal crops, vegetables and fruits. Specially, A. alternata generates toxins which contaminate food and feed. To date, management of A. alternata relies primarily on fungicides. However, the control efficacy in most cases is below expectation due to ubiquity of A. alternata and resistance to fungicides. To mitigate resistance and develop long-lasting fungicides, uncovering multiple rather than single target is a prerequisite. Membrane proteins are potential targets of fungicides owing to wide participation in myriad biochemical events especially material transport, signal transduction and pathogenicity. However, so far, little is known about the distribution and molecular structure of A. alternata membrane proteins (AAMPs). Herein we summarize AAMPs by data mining and subsequent structure prediction. We also outline the state-of-the-art research advances of AAMPs especially those closely related to pathogenicity. Overall, this review aims to portray a picture of AAMPs and provide valuable insights for future development of highly efficient fungicides towards A. alternata or beyond.

Development of orthogonal T7 expression system in Klebsiella pneumoniae.

Most expression systems are tailored for model organisms rather than nonmodel organisms. However, heterologous gene expression in model organisms constrains the innate advantages of original strain carrying gene of interest. In this study, T7 expression system was developed in nonmodel bacterium Klebsiella pneumoniae for production of chemicals. First, we engineered a recombinant K. pneumoniae strain harboring two vectors. One vector was used to express T7 RNA polymerase (T7 RNAP) which would drive the expression of egfp in the other vector. This recombinant strain demonstrated 15.73-fold of fluorescence relative to wild-type K. pneumoniae and showed similar level of fluorescence to recombinant Escherichia coli overexpressing egfp. When egfp was replaced by puuC, an endogenous aldehyde dehydrogenase catalyzing 3-hydroxypropionic acid (3-HP) biosynthesis in K. pneumoniae, the recombinant strain coexpressing T7 RNAP and PuuC showed high-level PuuC expression. In shake-flask cultivation, this recombinant strain produced 1.72 g/L 3-HP in 24 hr, which was 3.24 times that of wild-type K. pneumoniae (0.53 g/L). To mitigate plasmid burden, the vector expressing T7 RNAP was eliminated, but the T7 RNAP expression cassette was integrated into K. pneumoniae genome. The resulting strain harboring only PuuC expression vector produced 2.44 g/L 3-HP in 24 hr under shake-flask conditions, which was 1.46 times that of the strain harboring both T7 RNAP and PuuC expression vectors. In bioreactor cultivation, this strain generated 67.59 g/L 3-HP and did not show significantly halted growth. Overall, these results indicate that the engineered T7 expression system functioned efficiently in K. pneumoniae. This study provides a paradigm for the development of T7 expression system in prokaryotes.

Improved Production of Pyrroloquinoline Quinone by Simultaneous Augmentation of Its Synthesis Gene Expression and Glucose Metabolism in Klebsiella pneumoniae.

Klebsiella pneumoniae can naturally synthesize pyrroloquinoline quinone (PQQ), but current low yield restricts its commercialization. Here, we reported that PQQ production can be improved by simultaneously intensifying PQQ gene expression and glucose metabolism. Firstly, tandem repetitive tac promoters were constructed to overexpress PQQ synthesis genes. Results showed that when three repeats of tac promoter were recruited to overexpress PQQ synthesis genes, the recombinant strain generated 1.5-fold PQQ relative to the strain recruiting only one tac promoter. Quantitative real-time PCR (qRT-PCR) revealed the increased transcription levels of PQQ synthesis genes. Next, fermentation parameters were optimized to augment the glucose direct oxidation pathway (GDOP) mediated by PQQ-dependent glucose dehydrogenase (PQQ-GDH). Results demonstrated that the cultivation conditions of sufficient glucose (≥ 32 g/L), low pH (5.8), and limited potassium (0.7 nmol/L) significantly promoted the biosynthesis of gluconic acid, 2-ketogluconic acid, and PQQ. In optimum shake flask fermentation conditions, the K. pneumoniae strain overexpressing PQQ synthesis genes under three repeats of tac promoter generated 363.3 nmol/L of PQQ, which was 2.6-fold of that in original culture conditions. In bioreactor cultivation, this strain produced 2371.7 nmol/L of PQQ. To our knowledge, this is the highest PQQ titer reported so far using K. pneumoniae as a host strain. Overall, simultaneous intensification of pqq gene expression and glucose metabolism is effective to improve PQQ production.

Engineering a CRISPR Interference System To Repress a Class 1 Integron in Escherichia coli.

Microbial multidrug resistance (MDR) poses a huge threat to human health. Bacterial acquisition of MDR relies primarily on class 1 integron-involved horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). To date, no strategies other than the use of antibiotics can efficiently cope with MDR. Here, we report that an engineered CRISPR interference (CRISPRi) system can markedly reduce MDR by blocking a class 1 integron in Escherichia coli Using CRISPRi to block plasmid R388 class 1 integron, E. coli recombinants showed halted growth upon exposure to relevant antibiotics. A microplate alamarBlue assay showed that both subgenomic RNAs (sgRNAs) R3 and R6 led to 8- and 32-fold decreases in half-maximal inhibitory concentrations (IC50) for trimethoprim and sulfamethoxazole, respectively. Reverse transcription and quantitative PCR (RT-qPCR) revealed that the strain employing sgRNA R6 exhibited 97% and 84% decreases in the transcriptional levels of the dfrB2 cassette and sul1, two typical ARGs, respectively. RT-qPCR analysis also demonstrated that the strain recruiting sgRNA R3 showed a 96% decrease in the transcriptional level of intI1, and a conjugation assay revealed a 1,000-fold decrease in HGT rates of ARGs. Overall, the sgRNA R3 targeting the 31 bp downstream of the Pc promoter on the intI1 nontemplate strand outperformed other sgRNAs in reducing integron activity. Furthermore, this CRISPRi system is reversible, genetically stable, and titratable by varying the concentration of the inducer. To our knowledge, this is the first report on exploiting a CRISPRi system to reduce the class 1 integron in E. coli This study provides valuable insights for future development of CRISPRi-based antimicrobial agents and cellular therapy to suppress MDR.

Physiological investigations of the influences of byproduct pathways on 3-hydroxypropionic acid production in Klebsiella pneumoniae.

Klebsiella pneumoniae can naturally synthesize 3-hydroxypropionic acid (3-HP), 1,3-propanediol (1,3-PD), and 2,3-butanediol (2,3-BD) from glycerol. However, biosynthesis of these industrially important chemicals is constrained by troublesome byproducts. To clarify the influences of byproducts on 3-HP production, in this study, a total of eight byproduct-producing enzyme genes including pmd, poxB, frdB, fumC, dhaT, ilvH, adhP, and pflB were individually deleted from the K. pneumoniae genome. The resultant eight mutants presented different levels of metabolites. In 24-h shake-flask cultivation, the adhP- and pflB-deletion mutants produced 0.41 and 0.44 g/L 3-HP, respectively. Notably, the adhP and pflB double deletion mutant K. pneumoniaeΔadhPΔpflB produced 1.58 g/L 3-HP in 24-h shake-flask cultivation. When K. pneumoniaeΔadhPΔpflB was harnessed as a host strain to overexpress PuuC, a native aldehyde dehydrogenase (ALDH) catalyzing 3-hydroxypropionaldehyde (3-HPA) to 3-HP, the resulting recombinant strain K. pneumoniaeΔadhPΔpflB(pTAC-puuC) (pTAC-puuC is PuuC expression vector) generated 66.91 g/L 3-HP with a cumulative yield of 70.84% on glycerol in 60-h bioreactor cultivation. Additionally, this strain showed 2.3-, 5.1-, and 0.67-fold decrease in the concentrations of 1,3-PD, 2,3-BD, and acetic acid compared with the reference strain K. pneumoniae(pTAC-puuC). These results indicated that the byproducts exerted differential impacts on the production of 3-HP, 1,3-PD, and 2,3-BD. Although combinatorial elimination of byproduct pathways could reprogram glycerol flux, the enzyme 1,3-propanediol oxidoreductase (DhaT) that catalyzes 3-HPA to 1,3-PD and the enzymes ALDHs, especially, PuuC are most pivotal for 3-HP production. This study provides a deep understanding of how byproducts affect the production of 3-HP, 1,3-PD, and 2,3-BD in K. pneumoniae.

Exploiting tandem repetitive promoters for high-level production of 3-hydroxypropionic acid.

High-level biosynthesis of desired metabolites is challenging due to complexity of metabolic networks. Here, we report that platform chemical 3-hydroxypropionic acid (3-HP) can be overproduced through promoter engineering and growth-sustaining cultivation, two parallel strategies relying on RNA polymerases (RNAPs). First, we screened a promoter library and revealed that IPTG-inducible tac promoter was most effective for overexpression of PuuC, an endogenous aldehyde dehydrogenase catalyzing 3-HP biosynthesis in Klebsiella pneumoniae. Next, tandem repetitive tac promoters were harnessed to accommodate adequate RNAPs. When three tandem repetitive tac promoters were recruited to overexpress PuuC, up to 102.61 g/L 3-HP was produced. This is the highest 3-HP titer reported so far. In addition, lactic acid completely vanished during the late stage of fermentation. The backflow of lactic acid to pyruvic acid saves the trouble of downstream separation of lactic acid from 3-HP, which has long been a mission impossible because they are small-molecule isomers. Furthermore, timely removal of acid stress and replenishment of nitrogen source are crucial for 3-HP biosynthesis. A mathematical model shows that RNAPs modulate the tradeoff between bacterial growth and 3-HP formation. Overall, promoter engineering and growth-promoting cultivation jointly leverage RNAPs to maximize 3-HP. This study provides a paradigm for maximizing growth-coupled metabolites.

Engineering CRISPR interference system in Klebsiella pneumoniae for attenuating lactic acid synthesis.

BACKGROUND: Klebsiella pneumoniae is a promising industrial species for bioproduction of bulk chemicals such as 1,3-propanediol, 2,3-butanediol and 3-hydroxypropionic acid (3-HP). However, lactic acid is a troublesome by-product when optimizing for 3-HP production. Therefore, it is highly desirable to minimize lactic acid. RESULTS: Here, we show that lactic acid synthesis can be largely blocked by an engineered CRISPR interference (CRISPRi) system in K. pneumoniae. EGFP was recruited as a reporter of this CRISPRi system. Fluorescence assay of this CRISPRi system showed that enhanced green fluorescent protein (EGFP) expression level was repressed by 85-90%. To further test this CRISPRi system, guide RNAs were designed to individually or simultaneously target four lactate-producing enzyme genes. Results showed that all lactate-producing enzyme genes were significantly repressed. Notably, D-lactate dehydrogenase (ldhA) was shown to be the most influential enzyme for lactic acid formation in micro-aerobic conditions, as inhibiting ldhA alone led to lactic acid level similar to simultaneously repressing four genes. In shake flask cultivation, the strain coexpressing puuC (an aldehyde dehydrogenase catalyzing 3-hydroxypropionaldehyde to 3-HP) and dCas9-sgRNA inhibiting ldhA produced 1.37-fold 3-HP relative to the reference strain. Furthermore, in bioreactor cultivation, this CRISPRi strain inhibiting ldhA produced 36.7 g/L 3-HP, but only generated 1 g/L lactic acid. Clearly, this engineered CRISPRi system largely simplified downstream separation of 3-HP from its isomer lactic acid, an extreme challenge for 3-HP bioprocess. CONCLUSIONS: This study offers a deep understanding of lactic acid metabolism in diverse species, and we believe that this CRISPRi system will facilitate biomanufacturing and functional genome studies of K. pneumoniae or beyond.

Development of cyclic AMP receptor protein-based artificial transcription factor for intensifying gene expression.

Vector-dependent gene overexpression typically relies on an efficient operon and sufficient RNA polymerases (RNAPs). The lac (lactose) operon is a paradigm of transcription control, and cyclic AMP receptor protein (CRP) is a global regulator capable of recruiting RNAPs. However, the gap between lac operon and CRP has not been well bridged. In this work, CRP was fused to lac repressor protein (lacI) to form an artificial transcription factor (ATF) with the expectation that when LacI acted on the lacO-positioned upstream of gene of interest, the LacI-tethered CRP would trap RNAPs and thus improve the expression of PuuC, an aldehyde dehydrogenase catalyzing 3-hydroxypropionaldehyde (3-HPA) to 3-hydroxypropionic acid (3-HP) in Klebsiella pneumoniae. As expected, SDS-PAGE and HPLC showed enhanced PuuC expression and 3-HP production, respectively, compared to the control strain without expressing chimeric protein LacI-CRP. Moreover, quantitative real-time PCR demonstrated increased transcription levels of both PuuC and RNAP coding genes. In shake-flask cultivation, the recombinant K. pneumoniae strain coexpressing LacI-CRP and PuuC produced 1.67-fold of 3-HP relative to the stain only overexpressing PuuC. In bioreactor cultivation, the strain coexpressing LacI-CRP and PuuC produced 35.1 g/L 3-HP, whereas the strain without expressing LacI-CRP generated only 9.8 g/L 3-HP. Overall, these results indicated that as an ATF, LacI-CRP significantly boosted PuuC expression and 3-HP production. We envision that LacI-CRP as a plug-and-play part can be used for regulating gene expression.