Dynamic Control Strategy to Produce Riboflavin with Lignocellulose Hydrolysate in the Thermophile Geobacillus thermoglucosidasius.

Efficient utilization of both glucose and xylose, the two most abundant sugars in biomass hydrolysates, is one of the main objectives of biofermentation with lignocellulosic materials. The utilization of xylose is commonly inhibited by glucose, which is known as glucose catabolite repression (GCR). Here, we report a GCR-based dynamic control (GCR-DC) strategy aiming at better co-utilization of glucose and xylose, by decoupling the cell growth and biosynthesis of riboflavin as a product. Using the thermophilic strain Geobacillus thermoglucosidasius DSM 2542 as a host, we constructed additional riboflavin biosynthetic pathways that were activated by xylose but not glucose. The engineered strains showed a two-stage fermentation process. In the first stage, glucose was preferentially used for cell growth and no production of riboflavin was observed, while in the second stage where glucose was nearly depleted, xylose was effectively utilized for riboflavin biosynthesis. Using corn cob hydrolysate as a carbon source, the optimized riboflavin yields of strains DSM2542-DCall-MSS (full pathway dynamic control strategy) and DSM2542-DCrib (single-module dynamic control strategy) were 5.3- and 2.3-fold higher than that of the control strain DSM 2542 Rib-Gtg constitutively producing riboflavin, respectively. This GCR-DC strategy should also be applicable to the construction of cell factories that can efficiently use natural carbon sources with multiple sugar components for the production of high-value chemicals in future.

Correction to: Heterologous production of chlortetracycline in an industrial grade Streptomyces rimosus host.

The original version of this article contains an error.

Heterologous production of chlortetracycline in an industrial grade Streptomyces rimosus host.

High-yielding industrial Streptomyces producer is usually obtained by multiple rounds of random mutagenesis and screening. These strains have great potential to be developed as the versatile chassis for the discovery and titer improvement of desired heterologous products. Here, the industrial strain Streptomyces rimosus 461, which is a high producer of oxytetracycline, has been engineered as a robust host for heterologous expression of chlortetracycline (CTC) biosynthetic gene cluster. First, the industrial chassis strain SR0 was constructed by deleting the whole oxytetracycline gene cluster of S. rimosus 461. Then, the biosynthetic gene cluster ctc of Streptomyces aureofaciens ATCC 10762 was integrated into the chromosome of SR0. With an additional constitutively expressed cluster-situated activator gene ctcB, the CTC titer of the engineering strain SRC1 immediately reached 1.51 g/L in shaking flask. Then, the CTC titers were upgraded to 2.15 and 3.27 g/L, respectively, in the engineering strains SRC2 and SRC3 with the enhanced ctcB expression. Further, two cluster-situated resistance genes were co-overexpressed with ctcB. The resultant strain produced CTC up to 3.80 g/L in shaking flask fermentation, which represents 38 times increase in comparison with that of the original producer. Overall, SR0 presented in this study have great potential to be used for heterologous production of tetracyclines and other type II polyketides.

Improvement of stress tolerance and riboflavin production of Bacillus subtilis by introduction of heat shock proteins from thermophilic bacillus strains.

In this study, stress tolerance devices consisting of heat shock protein (HSP) genes from thermophiles Geobacillus and Parageobacillus were introduced into riboflavin-producing strain Bacillus subtilis 446 to improve its stress tolerance and riboflavin production. The 12 HSP homologs were selected from 28 Geobacillus and Parageobacillus genomes according to their sequence clustering and phylogenetically analysis which represents the diversity of HSPs from thermophilic bacillus. The 12 HSP genes and 2 combinations of them (PtdnaK-PtdnaJ-PtgrpE and PtgroeL-PtgroeS) were heterologously expressed in B. subtilis 446 under the control of a strong constitutive promoter P43. Most of the 14 engineered strains showed increased cell density at 44 to 48 °C and less cell death at 50 °C compared with the control strains. Among them, strains B.s446-HSP20-3, B.s446-HSP20-2, and B.s446-PtDnaK-PtDnaJ-PtGrpE increased their cell densities over 25% at 44 to 48 °C. They also showed 5-, 4-, and 4-fold improved cell survivals after the 10-h heat shock treatment at 50 °C, respectively. These three strains also showed reduced cell death rates under osmotic stress of 10% NaCl, indicating that the introduction of HSPs improved not only the heat tolerance of B. subtilis 446 but also its osmotic tolerance. Fermentation of these three strains at higher temperatures of 39 and 43 °C showed 23-66% improved riboflavin titers, as well as 24-h shortened fermentation period. These results indicated that implanting HSPs from thermophiles to B. subtilis 446 would be an efficient approach to improve its stress tolerance and riboflavin production.

Improvement of oxytetracycline production mediated via cooperation of resistance genes in Streptomyces rimosus.

Increasing the self-resistance levels of Streptomyces is an effective strategy to improve the production of antibiotics. To increase the oxytetracycline (OTC) production in Streptomyces rimosus, we investigated the cooperative effect of three co-overexpressing OTC resistance genes: one gene encodes a ribosomal protection protein (otrA) and the other two express efflux proteins (otrB and otrC). Results indicated that combinational overexpression of otrA, otrB, and otrC (MKABC) exerted a synergetic effect. OTC production increased by 179% in the recombinant strain compared with that of the wild-type strain M4018. The resistance level to OTC was increased by approximately two-fold relative to the parental strain, thereby indicating that applying the cooperative effect of self-resistance genes is useful to improve OTC production. Furthermore, the previously identified cluster-situated activator OtcR was overexpressed in MKABC in constructing the recombinant strain MKRABC; such strain can produce OTC of approximately 7.49 g L-1, which represents an increase of 19% in comparison with that of the OtcR-overexpressing strain alone. Our work showed that the cooperative overexpression of self-resistance genes is a promising strategy to enhance the antibiotics production in Streptomyces.

Heterologous expression of oxytetracycline biosynthetic gene cluster in Streptomyces venezuelae WVR2006 to improve production level and to alter fermentation process.

Heterologous expression is an important strategy to activate biosynthetic gene clusters of secondary metabolites. Here, it is employed to activate and manipulate the oxytetracycline (OTC) gene cluster and to alter OTC fermentation process. To achieve these goals, a fast-growing heterologous host Streptomyces venezuelae WVR2006 was rationally selected among several potential hosts. It shows rapid and dispersed growth and intrinsic high resistance to OTC. By manipulating the expression of two cluster-situated regulators (CSR) OtcR and OtrR and precursor supply, the OTC production level was significantly increased in this heterologous host from 75 to 431 mg/l only in 48 h, a level comparable to the native producer Streptomyces rimosus M4018 in 8 days. This work shows that S. venezuelae WVR2006 is a promising chassis for the production of secondary metabolites, and the engineered heterologous OTC producer has the potential to completely alter the fermentation process of OTC production.

[High throughput screening atrazine chlorohydrolase mutants with enhanced activity through Haematococcus pluvialis expression system].

Developing a high-throughput screening method is of great importance for directed evolution of atrazine chlorohydrolase. A mutagenesis library of atzA from Pseudomonas sp. ADP and Arthrobacter sp. AD1 was constructed using error-prone PCR and DNA shuffling. Candidate mutants were screened through Haematococcus pluvialis expression system, using atrazine as selection pressure. Sequence analysis showed that mutations in the obtained 12 mutants with enhanced activity were all point-substitutions and scattered throughout the gene. Enzymatic activity analysis showed that the mutants all had higher activities than that of the wild type. The activities were 1.8-3.6 fold of the wild-type enzyme when cultured in BBM medium with 1 mg/L atrazine, whereas 1.8-2.6 fold with 2 mg/L atrazine. These results indicated that Haematococcus pluvialis expression system is an ideal high throughput screening system for directed evolution of atrazine chlorohydrolase.

Transgenic tobacco plants expressing atzA exhibit resistance and strong ability to degrade atrazine.

Atrazine chlorohydrolase (AtzA) catalyzes hydrolytic dechlorination and can be used in detoxification of atrazine, a herbicide widely employed in the control of broadleaf weeds. In this study, to investigate the potential use of transgenic tobacco plants for phytoremediation of atrazine, atzA genes from Pseudomonas sp. strain ADP and Arthrobacter strain AD1 were transferred into tobacco. Three and four transgenic lines, expressing atzA-ADP and atzA-AD1, respectively, were produced by Agrobacterium-mediated transformation. Molecular characterization including PCR, RT-PCR and Southern blot revealed that atzA was inserted into the tobacco genome and stably inherited by and expressed in the progenies. Seeds of the T(1) transgenic lines had a higher germination percentage and longer roots than the untransformed plants in the presence of 40-150 mg/l atrazine. The T(2) transgenic lines grew taller, gained more dry biomass, and had higher total chlorophyll content than the untransformed plants after growing in soil containing 1 or 2 mg/kg atrazine for 90 days. No atrazine residue remained in the soil in which the T(2) transgenic lines were grown (except 401), while, in the case of the untransformed plants, 0.91 mg (81.3%) and 1.66 mg (74.1%) of the atrazine still remained in the soil containing 1 and 2 mg/kg of atrazine, respectively, indicating that the transgenic lines could degrade atrazine effectively. The transgenic tobacco lines developed could be useful for phytoremediation of atrazine-contaminated soil and water.