Tacticity Characterization of Biosynthesized Polyhydroxyalkanoates Containing (S)- and (R)-3-Hydroxy-2-Methylpropionate Units.

Polyhydroxyalkanoates (PHAs), aliphatic polyesters synthesized by microorganisms, have gained considerable attention as biodegradable plastics. Recently, α-carbon-methylated PHAs have been shown to exhibit several interesting properties that differ from those of conventional PHAs, such as their crystallization behavior and material properties. This study investigated α-carbon methylated (S)- and (R)-3-hydroxy-2-methylpropionate (3H2MP) as new repeating units. 3H2MP units were homopolymerized or copolymerized with (R)-3-hydroxybutyrate (3HB) by manipulating the culture conditions of recombinant Escherichia coli LSBJ. Consequently, PHAs with 3H2MP units ranging from 5 to 100 mol % were synthesized by external addition of (R)- and (S)-enantiomers or the racemic form of 3H2MPNa. The (S)-3H2MP precursor supplemented into the culture medium was almost directly polymerized into PHA while maintaining its chirality. Therefore, a highly isotactic P(3H2MP) (R:S = 1:99) was synthesized, which displayed a melting temperature of 114-119 °C and a relatively high enthalpy of fusion (68 J/g). In contrast, in cultures supplemented with (R)-3H2MP, the precursor was racemized and polymerized into PHA, resulting in the synthesis of the amorphous polymer atactic P(3H2MP) (R:S = 40:60). However, racemization was not observed at a low concentration of the (R)-3H2MP precursor, thereby synthesizing P(3HB-co-8 mol % 3H2MP) with 100% (R)-3H2MP units. The thermogravimetric analysis revealed that the thermal degradation temperatures at 5% weight loss of P(3H2MP)s occurred at approximately 313 °C, independent of tacticity, which is substantially higher than that of P(3HB) (257 °C). This study demonstrates a new concept for controlling the physical properties of biosynthesized PHA by manipulating the polymers' tacticity using 3H2MP units.

Engineering the Tat-secretion pathway of Bacillus licheniformis for the secretion of cytoplasmic enzyme arginase.

The industrial bacterium Bacillus licheniformis has long been used as a microbial factory for the production of enzymes due to its ability to secrete copious amounts of native extracellular proteins and its generally regarded as safe (GRAS) status. However, most attempts to use B. licheniformis to produce heterologous and cytoplasmic enzymes primarily via the general secretory (Sec) pathway have had limited success. The twin-arginine transport (Tat) pathway offers a promising alternative for the extracellular export of Sec-incompatible proteins because it transports full, correctly folded proteins. However, compared to the Sec pathway, the yields of the Tat pathway have historically been too low for commercial use. To improve the export efficiency of the Tat pathway, we identified the optimal Tat-dependent signal peptides and increased the abundance of the Tat translocases, the signal peptidase (SPase), and the intracellular chaperones. These strategic modifications significantly improved the Tat-dependent secretion of the cytoplasmic enzyme arginase into the culture medium using B. licheniformis. The extracellular enzymatic activity of arginase showed a 5.2-fold increase after these modifications. Moreover, compared to the start strain B. licheniformis 0F3, the production of extracellular GFP was improved by 3.8 times using the strategic modified strain B. licheniformis 0F13, and the extracellular enzymatic activity of SOX had a 1.3-fold increase using the strain B. licheniformis 0F14. This Tat-based production chassis has the potential for enhanced production of Sec-incompatible enzymes, therefore expanding the capability of B. licheniformis as an efficient cellular factory for the production of high-value proteins. KEY POINTS: • Systematic genetic modification of Tat-pathway in B. licheniformis. • Significant enhancement of the secretion capacity of Tat pathway for delivery the cytoplasmic enzyme arginase. • A new platform for efficient extracellular production of Sec-incompatible enzymes.