Enhanced Production of Bacterial Cellulose in Komagataeibacter xylinus Via Tuning of Biosynthesis Genes with Synthetic RBS.

Bacterial cellulose (BC) has outstanding physical and chemical properties, including high crystallinity, moisture retention, and tensile strength. Currently, the major producer of BC is Komagataeibacter xylinus. However, due to limited tools of expression, this host is difficult to engineer metabolically to improve BC productivity. In this study, a regulated expression system for K. xylinus with synthetic ribosome binding site (RBS) was developed and used to engineer a BC biosynthesis pathway. A synthetic RBS library was constructed using green fluorescent protein (GFP) as a reporter, and three synthetic RBSs (R4, R15, and R6) with different strengths were successfully isolated by fluorescence-activated cell sorting (FACS). Using synthetic RBS, we optimized the expression of three homologous genes responsible for BC production, pgm, galU, and ndp, and thereby greatly increased it under both static and shaking culture conditions. The final titer of BC under static and shaking conditions was 5.28 and 3.67 g/l, respectively. Our findings demonstrate that reinforced metabolic flux towards BC through quantitative gene expression represents a practical strategy for the improvement of BC productivity.

Development and characterization of a Nannochloropsis mutant with simultaneously enhanced growth and lipid production.

BACKGROUND: The necessity to develop high lipid-producing microalgae is emphasized for the commercialization of microalgal biomass, which is environmentally friendly and sustainable. Nannochloropsis are one of the best industrial microalgae and have been widely studied for their lipids, including high-value polyunsaturated fatty acids (PUFAs). Many reports on the genetic and biological engineering of Nannochloropsis to improve their growth and lipid contents have been published. RESULTS: We performed insertional mutagenesis in Nannochloropsis salina, and screened mutants with high lipid contents using fluorescence-activated cell sorting (FACS). We isolated a mutant, Mut68, which showed improved growth and a concomitant increase in lipid contents. Mut68 exhibited 53% faster growth rate and 34% higher fatty acid methyl ester (FAME) contents after incubation for 8 days, resulting in a 75% increase in FAME productivity compared to that in the wild type (WT). By sequencing the whole genome, we identified the disrupted gene in Mut68 that encoded trehalose-6-phosphate (T6P) synthase (TPS). TPS is composed of two domains: TPS domain and T6P phosphatase (TPP) domain, which catalyze the initial formation of T6P and dephosphorylation to trehalose, respectively. Mut68 was disrupted at the TPP domain in the C-terminal half, which was confirmed by metabolic analyses revealing a great reduction in the trehalose content in Mut68. Consistent with the unaffected N-terminal TPS domain, Mut68 showed moderate increase in T6P that is known for regulation of sugar metabolism, growth, and lipid biosynthesis. Interestingly, the metabolic analyses also revealed a significant increase in stress-related amino acids, including proline and glutamine, which may further contribute to the Mut68 phenotypes. CONCLUSION: We have successfully isolated an insertional mutant showing improved growth and lipid production. Moreover, we identified the disrupted gene encoding TPS. Consistent with the disrupted TPP domain, metabolic analyses revealed a moderate increase in T6P and greatly reduced trehalose. Herein, we provide an excellent proof of concept that the selection of insertional mutations via FACS can be employed for the isolation of mutants with improved growth and lipid production. In addition, trehalose and genes encoding TPS will provide novel targets for chemical and genetic engineering, in other microalgae and organisms as well as Nannochloropsis.

Efficient production of d-lactate from methane in a lactate-tolerant strain of Methylomonas sp. DH-1 generated by adaptive laboratory evolution.

BACKGROUND: Methane, a main component of natural gas and biogas, has gained much attention as an abundant and low-cost carbon source. Methanotrophs, which can use methane as a sole carbon and energy source, are promising hosts to produce value-added chemicals from methane, but their metabolic engineering is still challenging. In previous attempts to produce lactic acid (LA) from methane, LA production levels were limited in part due to LA toxicity. We solved this problem by generating an LA-tolerant strain, which also contributes to understanding novel LA tolerance mechanisms. RESULTS: In this study, we engineered a methanotroph strain Methylomonas sp. DH-1 to produce d-lactic acid (d-LA) from methane. LA toxicity is one of the limiting factors for high-level production of LA. Therefore, we first performed adaptive laboratory evolution of Methylomonas sp. DH-1, generating an LA-tolerant strain JHM80. Genome sequencing of JHM80 revealed the causal gene watR, encoding a LysR-type transcription factor, whose overexpression due to a 2-bp (TT) deletion in the promoter region is partly responsible for the LA tolerance of JHM80. Overexpression of the watR gene in wild-type strain also led to an increase in LA tolerance. When d form-specific lactate dehydrogenase gene from Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 was introduced into the genome while deleting the glgA gene encoding glycogen synthase, JHM80 produced about 7.5-fold higher level of d-LA from methane than wild type, suggesting that LA tolerance is a critical limiting factor for LA production in this host. d-LA production was further enhanced by optimization of the medium, resulting in a titer of 1.19 g/L and a yield of 0.245 g/g CH4. CONCLUSIONS: JHM80, an LA-tolerant strain of Methylomonas sp. DH-1, generated by adaptive laboratory evolution was effective in LA production from methane. Characterization of the mutated genes in JHM80 revealed that overexpression of the watR gene, encoding a LysR-type transcription factor, is responsible for LA tolerance. By introducing a heterologous lactate dehydrogenase gene into the genome of JHM80 strain while deleting the glgA gene, high d-LA production titer and yield were achieved from methane.

Selective bio-oxidation of propane to acetone using methane-oxidizing Methylomonas sp. DH-1.

Propane is the major component of liquefied petroleum gas (LPG). Nowadays, the use of LPG is decreasing, and thus utilization of propane as a chemical feedstock is in need of development. An efficient biological conversion of propane to acetone using a methanotrophic whole cell as the biocatalyst was proposed and investigated. A bio-oxidation pathway of propane to acetone in Methylomonas sp. DH-1 was analyzed by gene expression profiling via RNA sequencing. Propane was oxidized to 2-propanol by particulate methane monooxygenase and subsequently to acetone by methanol dehydrogenases. Methylomonas sp. DH-1 was deficient in acetone-converting enzymes and thus accumulated acetone in the absence of any enzyme inhibition. The maximum accumulation, average productivity and specific productivity of acetone were 16.62 mM, 0.678 mM/h and 0.141 mmol/g cell/h, respectively, under the optimized conditions. Our study demonstrates a novel method for the bioconversion of propane to acetone using methanotrophs under mild reaction condition.

Batch conversion of methane to methanol using Methylosinus trichosporium OB3b as biocatalyst.

Recently, methane has attracted much attention as an alternative carbon feedstock since it is the major component of abundant shale and natural gas. In this work, we produced methanol from methane using whole cells of Methylosinus trichosporium OB3b as the biocatalyst. M. trichosporium OB3b was cultured on NMS medium with a supply of 7:3 air/methane ratio at 30°C. The optimal concentrations of various methanol dehydrogenase inhibitors such as potassium phosphate and EDTA were determined to be 100 and 0.5 mM, respectively, for an efficient production of methanol. Sodium formate (40 mM) as a reducing power source was added to enhance the conversion efficiency. A productivity of 49.0 mg/l·h, titer of 0.393 g methanol/l, and conversion of 73.8% (mol methanol/mol methane) were obtained under the optimized batch condition.